Abstract:

A novel method for producing a bone filling material is provided. The
method comprises the steps of: (a) kneading ingredient comprising
calcium-based material and material comprising binder; (b) molding a
predetermined shape of the mixture obtained in step (a) with an injection
molding machine having a mold; (c) removing the binder contained in the
mold formed in step (b) (i.e., degreasing) to obtain a degreased body;
(d) and heating and sintering the degreased body obtained in step (c) to
obtain a sintered body.

Claims:

1.-22. (canceled)

23. A bone filling material comprising calcium-based materials, the bone
filling material comprising a plurality of protruding parts:characterized
in that thrombin is impregnated, administered or powder blended on the
surface of the protruding parts.

24.-26. (canceled)

Description:

BACKGROUND OF THE INVENTION

[0001]1. Field of the Invention

[0002]The present invention relates to a bone filling material which is
produced applying the powder injection molding method, and a method for
producing the bone filling material and a three-dimensional cell culture
carrier. In particular, the present invention relates to a method for
producing a minute bone filling material having a plurality of protruding
parts, a bone filling material, and a three-dimensional cell culture
carrier.

[0003]2. Description of the Related Art

[0004]In the conventional therapeutic technique, when a part of bone
tissue is lost in an accident or in a surgical treatment, regeneration of
the lost bone was promoted, for example, by filling the lost part of bone
with bone filling material which replaces the lost bone tissue. Calcium
phosphate-based material such as hydroxyapatite and β-TCP, or a
biodegradable plastic such as polylactic acid was, for example, used as
ingredients of the bone filling material.

[0005]A tetrapod-shaped bone filling material (bone fixing material) is
disclosed in Japanese Patent Laid-Open No. 2004-97259 (e.g., FIG. 1 to
FIG. 3 of the bulletin). Note that "tetrapod" is a trademark. And a
method of melt molding using a split mold is disclosed (in paragraph
[0014] and [0025] of the bulletin) as a method for producing
Tetrapod®-shaped bone filling material. However, the bone filling
material produced by the method of melt molding using a split mold does
not always have enough strength. And when calcium phosphate-based
material and biodegradable polymers were produced by the melt molding,
there is also a problem that the dimensional accuracy and the molding
density of a resultant bone filling material become uneven. Uneven
surface condition of the bone filling material is another problem. In
addition, the production method of the bone filling material disclosed in
the bulletin is not suitable for mass production.

[0006]A method for producing a rod-shaped or sheet-shaped bone filling
material using polylactic acid or hydroxyapatite is disclosed in Japanese
Patent Laid-Open No. 11-206871 (paragraph [0018]), wherein an injection
molded bone filling material is uniaxially extended. However the
resultant bone filling material is a polymer molecule such as polylactic
acid or a mixture of the polymer molecule and calcium phosphate-based
material. And the embodiment of the bulletin discloses the production of
a sheet-formed bone filling material by extrusion molding (paragraph
[0023]), but a complex-shaped (e.g., tetrapod-shaped) bone filling
material cannot be produced by extrusion molding.

[0007]Also, the powder injection molding method was used to produce metal
parts and ceramics. However, in the powder injection molding method,
material with poor biocompatibility is used as binder. So there was no
idea of molding a material which is embedded in vivo for the purpose of
tissue replacement, by the powder injection molding method. Besides the
bone filling material has to have bone tissue replacement ability in
vivo. So when a bone filling material is molded from powders such as
calcium-based materials by the powder injection molding method, a
preferred bone filling material cannot be obtained.

[0008]The object of the present invention is to provide a novel method for
producing bone filling material.

[0009]The object of the present invention is to provide a method for
producing bone filling material with less variation in shape and density.

[0010]The object of the present invention is to provide a method for
producing bone filling material which is easy to be removed from a mold
when it is injection molded.

[0011]The object of the present invention is to provide bone filling
material having a predetermined strength and a method for producing the
same.

[0012]The object of the present invention is to provide a bone filling
material having a predetermined pharmacological effect and a method for
producing the same.

[0013]Cell culture has been so far performed two-dimensionally. In
contrast, the object of the present invention is to provide a carrier
which can perform cell culture three-dimensionally.

[0014]The object of the present invention is to provide usages of the
above mentioned bone filling material which have not been considered so
far. In particular, usages of a three-dimensional cell culture carrier, a
separating carrier for chromatography, and the like is provided.

SUMMARY OF THE INVENTION

[0015]A bone filling material with a complicated minute shape cannot be
produced by the ordinary powder injection molding method, because, for
example, the molded body adheres to a fixed mold. This invention is made
preferably for the purpose of mass production of the bone filling
material having preferred physical properties. The present invention is
based on the following idea. That is, a bone filling material having
physical properties, such as less variation in surface area and density,
and appropriate level of hardness as a bone filling material, can be
produced by applying the powder injection molding method, which has been
used basically for manufacturing metal parts or ceramics, to a production
method of a bone filling material using calcium-based material such as
calcium phosphate-based material as ingredient powders ingeniously. Since
each of the bone filling material has even surface area and density,
appropriate dosage of pharmaceutical agent can be administered, even when
the pharmaceutical agent is incorporated in the bone filling material. In
addition, since each of the bone filling material is uniform in size, the
whole of bone filling materials can obtain porosity while maintaining the
intensity of each bone filling material. The present invention is also
based on the following idea. The resultant molded body can be prevented
from adhering to a fixed mold by using a mold and materials of the
present invention.

[0016]The method for producing a bone filling material of the present
invention basically comprises the steps of: (a) kneading ingredient
comprising calcium-based material and material comprising binder; (b)
molding a molded body having a predetermined shape from a kneaded
material obtained in step (a) with an injection molding machine having a
mold; (c) removing the binder contained in the molded body (i.e.,
degreasing) to obtain a degreased body, the molded body being obtained in
step (b); and (d) heating and sintering the degreased body to obtain a
sintered body, the degreased body being obtained in step (c).

[0017]A method for producing a bone filling material according to an
aspect of the present invention is the above described method, wherein
the calcium-based material comprises one or both of calcium
phosphate-based material and calcium carbonate-based material, and
wherein the calcium phosphate-based material is one or more than one kind
of hydroxyl apatite, carbonate apatite, fluorapatite, chlorapatite,
β-TCP, α-TCP, calcium metaphosphate, tetra calcium phosphate,
calcium hydrogen phosphate, octa calcium phosphate, calcium dihydrogen
phosphate, calcium pyrophosphate, the salts thereof, and the solvates
thereof. These materials effectively replace with osteocytes. So a bone
filling material contributing to bone regeneration can be provided.

[0018]The bone filling material obtained by the production method of the
present invention is generally minute, and need to have appropriate
physical properties as a bone filling material. So a preferred aspect of
the production method of the present invention produces a preferred bone
filling material by using a certain kind and amount of binder added to
ingredients. A specific example of the production method is one of the
above described methods for producing a bone filling material, wherein
the binder comprises (meta) acrylic-based resin, wax lubricant, and
lubricant. Another example of the production method is one of the above
described methods for producing a bone filling material, wherein the
binder comprises wax lubricant, and wherein the wax lubricant comprises
wax having melting point of 40° C. to 100° C. Since wax
lubricant having low melting point is used, the bone filling material can
be taken out of a mold easily, which enables effective production of the
bone filling material. A preferred embodiment of the present invention is
one of the above described methods for producing a bone filling material
wherein the binder comprises (meta) acrylic-based resin, ethylene-vinyl
acetate copolymer, paraffin wax, stearic acid and dibutyl phthalate, and
wherein the step (a) comprises the steps of: putting the (meta)
acrylic-based resin and the ethylene-vinyl acetate copolymer in a
kneading machine; putting the ingredient, the paraffin wax, and the
stearic acid in the kneading machine while kneading the (meta)
acrylic-based resin and the ethylene-vinyl acetate copolymer; and putting
the dibutyl phthalate in the kneading machine while kneading the (meta)
acrylic-based resin, the ethylene-vinyl acetate copolymer, the paraffin
wax, and the stearic acid. Although producing a bone filling material is
not easy, the most desirable bone filling material can be produced by
using particular resin.

[0019]A method for producing a bone filling material according to a
certain aspect of the present invention is one of the above described
methods for producing a bone filling material wherein the material
comprising binder further comprising glass components. If the strength of
a bone filling material can be controlled, the application of the bone
filling material will be extended. And the bone filling material
including glass components can achieve high strength.

[0020]A method for producing a bone filling material according to an
aspect of the present invention is one of the above described methods for
producing a bone filling material wherein the material comprising binder
further comprising salt or sugar. The bone filling material of the
present invention is to culture cells such as bone cells in vivo or in
vitro. So it is desired that the bone filling material have a surface to
which cells can be attached easily. And if the bone filling material is
produced by using ingredients including salt or sugar, a porous bone
filling material can be obtained by removing salt or sugar with water.
Then, pores where salts or sugars were embedded appear, and a preferred
physical structure for cellular culture can be obtained.

[0021]A method for producing a bone filling material according to an
aspect of the present invention is one of the above described methods for
producing a bone filling material, wherein the bone filling material has
a plurality of protruding parts (e.g., more than four protruding parts),
and wherein the mold comprises: a fixed mold having an inlet where
material (e.g., kneaded material) is injected; a movable mold being
contacted with the fixed mold when the material is injected, the movable
mold being apart from the fixed mold after a molded body is formed,
wherein the inlet of the fixed mold is located at a tip part of one of
the protruding parts so that the material for injection molding is
injected from the tip part of the protruding part, wherein a parting face
of the fixed mold and the movable mold has an inclined surface, the
inclined surface inclines toward the inlet of the fixed mold from the
edge face to the center of the parting face, and wherein the movable mold
has dent parts for providing predetermined wedges to the movable mold.
Producing a bone filling material accompanies difficulties. Those who
skilled in the art think of producing the protruding parts independently
and then combining them together, instead of adopting a method of
injection molding. But since the bone filling material of the present
invention is minute, it is difficult to produce the protruding parts
separately and combine them together. The mold of the present invention
can produce an injection body having a plurality of protruding parts in
one injection molding operation. The mold of the present invention also
made it easy for a molded body to be taken out from the fixed mold, for
example, by providing a three-dimensional parting face, wherein the
parting face of the fixed mold and the movable mold has an inclined
surface toward the inlet from the edge to the center of the parting face.
For example, when a split mold having a flat parting face is used to mold
a molded body, the molded body is highly likely to be stuck in a fixed
mold, and it is difficult to remove the molded body from the fixed mold.
But the above described mold having inclined parting face lowered the
possibility that a molded body remains in a fixed mold.

[0022]A preferred embodiment of the present invention is the above
described methods for producing a bone filling material, wherein the
height of the wedge is 1 μm to 1×102 μm. If a molded
body remains in the fixed mold, bone filling material can not be
produced. The wedge part serves as a wedge to the moving part, which
prevents a molded body being left in the fixed mold when the mold is
opened.

[0023]A preferred embodiment of the present invention is one of the above
described methods for producing a bone filling material, wherein the
height of the wedge is 1 μm to 1×102 μm. If a molded
body remains in the fixed mold, bone filling material can not be
produced. The groove parts increase the surface area of the movable mold
part, thereby preventing a molded body from being left in the fixed mold
when the mold is opened.

[0024]A preferred embodiment of the present invention is one of the above
described methods for producing a bone filling material, wherein the bone
filling material has four protruding parts (preferably so-called a
tetrapod-shaped bone filling material wherein four protruding parts
extend toward each vertex of the regular tetrahedron), and wherein the
movable mold has an ejector pin located inside the protruding part. It is
very important for injection molding to remove a molded body from a mold.
In this embodiment, a protruding pin is arranged so that it is located
inside a protruding part (so that the pin is directed from the other end
of the tip of a protruding part through the center of the mold toward the
tip of the protruding part), so a molded body can be taken out
effectively.

[0025]A preferred embodiment of the present invention is one of the above
described methods for manufacturing a bone filling material wherein the
bone filling material has a plurality of protruding parts. And the mold
comprises: a fixed mold; a movable mold being contacted with the fixed
mold when material is injected, the movable mold being apart from the
fixed mold after a molded body is formed, wherein an inlet of the
material for injection molding is located at a parting face of the fixed
mold and the movable mold, the parting face of the fixed mold and the
movable mold has an inclined surface, the inclined surface inclines
toward the center of the bone filling material from the edge face to the
center of the parting face, and the movable mold has groove parts for
providing predetermined wedges to the movable mold. An injection body
having a plurality of protruding parts can be obtained in one injection
molding operation by an ingenious mold at the timing of injection
molding.

[0026]A method for producing a bone filling material according to an
aspect of the present invention is the above described method for
producing a bone filling material wherein the step (c) comprises a step
of heating-up at 1° C./hour to 3×102° C./hour
(preferably 10° C./hour to 2×102° C./hour) until
the temperature reaches a value in the range of 110° C. to
300° C., which is the range of a first maintaining period. A
binder removal step, for example, has several stages of heating-up period
and maintaining period in accordance with the pyrolysis temperature of
resin contained in binder. Since binder is removed through several
heating-up stages, pyrolysis can be effectively performed from the resin
having low pyrolysis temperature, thereby effectively removing binder. In
particular, effective pyrolysis of resin having low pyrolysis temperature
improves sintering performance. In the present invention, since
temperature is raised as above described, the resin having low pyrolysis
temperature can be effectively pyrolyzed.

[0027]A method for producing a bone filling material according to an
aspect of the present invention is one of the above described methods for
producing bone filling material further comprising the step of
impregnating or administering pharmaceutical agent to the sintered body
obtained in the sintering step. And a preferred embodiment of the method
for producing bone filling material of the present invention is one of
the above described methods for producing bone filling material wherein
the pharmaceutical agent comprises one or more than one kind of an
osteogenesis/chondrogenesis promoter (including chondrogenesis promoting
factor), a joint disease therapeutic agent, a preventive and/or
therapeutic agent for bone/cartilage disease, a bone-regenerating agent,
a bone resorption-suppressing substance, an angiogenesis promoter, an
antibacterial agent, an antibiotics, or an anticancer agent. The
conventional bone filling material was aimed merely at bone regeneration
by being administered to defective parts of bone tissues. The bone
filling material of the present invention, pharmaceutical agents being
administered thereto, can prevent infections of affected sites as well as
promote bone regeneration. Namely, the present invention can provide a
bone filling material comprising: a plurality of protruding parts,
wherein an adhesiveness-imparting agent is impregnated, administered or
powder blended on the surface of the protruding parts; and calcium-based
materials.

[0028]A method for producing a bone filling material according to an
aspect of the present invention is one of the above described methods for
producing a bone filling material comprising the step of impregnating or
administering an adhesiveness-imparting agent to the sintered body
obtained in step (d). The adhesiveness-imparting agent, for example, is
thrombin. Also, an embodiment of the present invention is one of the
above described methods for producing a bone filling material further
comprising the steps of preparing a composition including two kinds of
adhesiveness-imparting agents; obtaining a first bone filling material
group by impregnating a certain bone filling material group with a first
composition or by applying a first composition to a certain bone filling
material group; obtaining a second bone filling material group by
impregnating a certain bone filling material group with a second
composition or by applying a second composition to a certain bone filling
material group, wherein the bone filling material groups including the
first bone filling material group and the second bone filling material
group are used as the bone filling materials, and wherein the first
composition comprises: one or more than one kind of compound represented
by the below-described general formula (I) or (II), or a first compound
including 3 to 8 repeating units represented by the below-described
general formula (III); and a first diluent (or a carrier), and wherein
the second composition comprises: one or more than one kind of compound
represented by the below-described general formula (I) or (II), or a
second compound including 3 to 8 repeating units represented by the
below-described general formula (III); and a second diluent:

X1--(OCH2CH2)n--X2 (I)

[0029]wherein X1 and X2 are the same or different and each
represents --R1COONHS (where R1 is a C1-7 alkylene group),
--COR1COONHS (where R1 is a C1-7 alkylene group),
--NOCOR1--R2 (where R1 is a C1-7 alkylene group, and
R2 is a maleimide group), --R1NH2 (where R1 is a
C1-7 alkylene group), --R1SH (where R1 is a C1-7
alkylene group), or --CO2PhNO2 (where Ph is an o-, m-, or
p-phenylene group), and n is an integer of 80 to 1000.

##STR00001##

[0030]wherein XII-1 to XII-4 are the same or different and each
represents --R1COONHS (where R1 is a C1-7 alkylene group),
--COR1COONHS (where R1 is a C1-7 alkylene group),
--NOCOR1--R2 (where R1 is a C1-7 alkylene group, and
R2 is a maleimide group), --R1NH2 (where R1 is a
C1-7 alkylene group), --R1SH (where R1 is a C1-7
alkylene group), or --CO2PhNO2 (where Ph is an o-, m-, or
p-phenylene group), and nII-1 to nII-4 are the same or
different and each represents an integer of 20 to 250.

##STR00002##

[0031]wherein XIII represents --R1COONHS (where R1 is a
C1-7 alkylene group), --COR1COONHS, --NOCOR1--R2
(where R1 is a C1-7 alkylene group, and R2 is a maleimide
group), --R1NH2 (where R1 is a C1-7 alkylene group),
--R1SH (where R1 is a C1-7 alkylene group), or
--CO2PhNO2 (where Ph is an o-, m-, or p-phenylene group), and
nIII represents an integer of 10 to 150.

[0032]A more preferred embodiment of the above described method for
producing a bone filling material is a method, wherein the first compound
comprises one or more than one kind of compound represented by the
general formula (I) or (II), wherein X1, X2 or XII-1 to
XII-4 are the same or different, and each represents
--NOCOR1--R2 or --R1NH2, and wherein the second
compound comprises one or more than one kind of compound represented by
the general formula (I) or (II), wherein X1, X2 or XII-1
to XII-4 are the same or different, and each represents
--COR1COONHS, --R1SH, or --CO2PhNO2 (where Ph is an
o-, m-, or p-phenylene group).

[0033]A bone filling material is intended to be administered in vivo. And
it is desirable that a plurality of the bone filling materials be
combined with each other, thereby maintaining a steric structure. The
conventional bone filling material has a fragile steric structure which
exhibits low-strength. However, the above described
adhesiveness-imparting agent enhances the adhesiveness of the each bone
filling material and the adhesiveness of the bone filling material to
bone tissues, thereby maintaining the steric structure. When a
heat-resistant adhesiveness-imparting agent is used, the
adhesiveness-imparting agent may be mixed with ingredient powders so that
a bone filling material which is powder blended with the
adhesiveness-imparting agent may be obtained (in this case, the
adhesiveness-imparting agent exists on the surface of the bone filling
material, and when the adhesiveness-imparting agent on the surface is
replaced with bone tissues, a new surface having the other
adhesiveness-imparting agent appears, thereby maintaining the
adhesiveness of the bone filling material). Also, a powdered
adhesiveness-imparting agent may be sprayed on the surface of a molded
body or a sintered body. Furthermore, adhesiveness-imparting agents may
be added to the surface of the bone filling material by powder blending,
wherein a plurality of bone filling agents and powdered
adhesiveness-imparting agents are mixed together and then agitated as
needed. Namely, the present invention also provides a bone filling
material comprising: a plurality of protruding parts, wherein an
adhesiveness-imparting agent is impregnated, administered or powder
blended on the surface of the protruding parts; and calcium-based
materials.

[0034]A method for producing a bone filling material according to an
aspect of the present invention is the above described method for
producing a bone filling material, wherein the bone filling material has
four protruding parts, the four protruding parts extending from the
center of the regular tetrahedron form of the bone filling material
toward each vertex thereof. A bone filling material having this structure
can form preferred continuous holes, so cells and the like can be
cultured easily.

[0035]A usage example of a bone filling material according to an aspect of
the present invention is a three-dimensional cell culture carrier
comprising one of the above described bone filling materials including
calcium-based material, and plurality of protruding parts. A preferred
embodiment of the three-dimensional cell culture carrier comprises a bone
filling material, wherein a pharmaceutical agent or an
adhesiveness-imparting agent is impregnated, administered or powder
blended on the surface of the bone filling material. Conventionally, it
was intended that a cell culture was performed in a two-dimensional
environment, such as in a Petri dish etc. But the present invention
enables an in vitro cell culture to be performed in a three-dimensional
environment effectively.

[0036]A usage example of a bone filling material according to a certain
aspect of the present invention is a separating carrier for
chromatography comprising a bone filling material including a plurality
of protruding parts and containing calcium-based material. As
demonstrated in the example described below, the bone filling material of
the present invention has preferred sorbability, and the sorbability can
be controlled, for example, by adjusting voids of the bone filling
material. So a plurality of bone filling materials can be used as
separating carriers for chromatography.

[0037]In the present invention, the powder injection molding method,
adjusted in accordance with calcium phosphate-based material as
ingredient powders, was used for producing the bone filling material. So
the present invention can provide a method for producing bone filling
material having high form accuracy and less variation in the volume.

[0038]In the present invention, since a predetermined wax was used as
binder, a method for producing a bone filling material which is easy to
be removed from a mold can be provided.

[0039]As demonstrated in the example described below, the present
invention can provide a bone filling material having a predetermined
strength and a method for producing the same.

[0040]The bone filling material of the present invention, having been
administered a pharmaceutical agent on the surface of a molded body, can
act as a pharmaceutical agent having a predetermined pharmacological
effect. So the present invention can provide a method for producing a
bone filling material having a predetermined pharmacological effect and a
method for producing the same.

[0041]Cell culture has been so far performed two-dimensionally. But, in
contrast, as demonstrated in the example described below, the present
invention can provide a carrier which can perform cell culture
three-dimensionally by using a plurality of bone filling materials
effectively.

[0042]The present invention provides usage examples of the above mentioned
bone filling material which have not been considered so far. In
particular, the usage of a three-dimensional cell culture carrier, a
separating carrier for chromatography, and the like can be provided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0043]FIG. 1 is a conceptual diagram showing an example of a mold used in
the method for producing a bone filling material of the present
invention.

[0044]FIG. 2 is a conceptual diagram for explaining an example of a
tetrapod-shaped bone filling material. FIG. 2(A) is a side view, FIG.
2(B) is a top view, and FIG. 2(C) is a perspective view.

[0045]FIG. 3 is a conceptual diagram for explaining an example of a
tetrapod-shaped bone filling material with its top portion cut off. FIG.
3(A) and FIG. 3(B) are side views, FIG. 3(C) is a top view, and FIG. 3(D)
is a perspective view.

[0046]FIG. 4 is a conceptual diagram for explaining an example of a bone
filling material having three protruding parts. FIG. 4(A) is a
perspective view, and FIG. 4(B) is a top view.

[0047]FIG. 5 is a conceptual diagram for explaining an example of a
hemispherical bone filling material. FIG. 5(A) is a perspective view, and
FIG. 5(B) is a bottom view.

[0048]FIG. 6 is a conceptual diagram for explaining an example of a bone
filling material having one or a plurality of hollowed parts in the
undersurface part. FIG. 6(A) is a perspective view, and FIG. 6(B) is a
bottom view.

[0049]FIG. 7 is a conceptual diagram for explaining an example of a
double-headed-shaped bone filling material. FIG. 7(A) is a perspective
view, and FIG. 7(B) and FIG. 7(C) are side views.

[0050]FIG. 8 is a conceptual diagram for explaining an example of a bone
filling material having protruding parts on the center thereof FIG. 8(A)
is a perspective view, and FIG. 8(B) is a side view.

[0051]FIG. 9 is a conceptual diagram for explaining an example of a
cross-shaped bone filling material. FIG. 9(A) is a perspective view, FIG.
9(B) is a top view, FIG. 9(C) is a side view, and FIG. 9(D) is a bottom
view.

[0052]FIG. 10 is a conceptual diagram for explaining an example of a
nearly planar bone filling material. FIG. 10(A) is a perspective view,
FIG. 10(B) is a top view, and FIG. 10(C) is a side view.

[0053]FIG. 11 is a conceptual diagram for explaining an example of a bone
filling material having protruding parts on one or both ends of the plane
face.

[0054]FIG. 12 is a conceptual diagram for explaining an example of a bone
filling material having an inclined top surface. FIG. 12(A) is a
perspective view, and FIG. 12(B) is a side view.

[0055]FIG. 13 is a CAD generated drawing of a bone filling material.

[0056]FIG. 14 is a photograph, in place of a diagram, showing a bone
filling material obtained in Example 1.

[0057]FIG. 15 is an electron microgram, in place of a diagram, showing a
bone filling material obtained in Example 1.

[0058]FIG. 16 is an electron microgram, in place of a diagram, showing
clustered bone filling materials obtained in Example 1.

[0059]FIG. 17 shows a relationship between the sintering temperature and
the bending strength of the bone filling material obtained in Example 1.

[0060]FIG. 18 are CT images by micro X-rays, in place of a diagram,
showing gatherings of the bone filling materials obtained in Example 1
and the existing artificial bone product. FIG. 18(A) shows gatherings of
the bone filling materials of the present invention. FIG. 18(B) shows
gatherings of the conventional artificial bone granules. FIG. 18(C) shows
the bone filling materials of the present invention filled in a cryotube.
FIG. 18(D) shows the existing artificial bone products filled in a
cryotube.

[0061]FIG. 19(A) to (F) are photographs, in place of diagrams, examining
cellular adhesiveness of a bone filling material of the present
invention. FIG. 19 (A) is a photograph showing a bone filling material
without cultured cells after four days culture. FIG. 19 (B) is a
photograph, after four days culture, showing a bone filling material on
which osteoblast-like cell lines MC3T3 were cultured. FIG. 19 (C) is a
photograph showing a bone filling material without cultured cells which
is Alkaline Phosphatase (ALP) stained after six days culture. FIG. 19 (D)
is a photograph, Alkaline Phosphatase (ALP) stained after six days
culture, showing a bone filling material on which osteoblast-like cell
lines MC3T3 were cultured. FIG. 19 (E) is a photograph showing a bone
filling material without cultured cells which is Alkaline Phosphatase
(ALP) stained after ten days culture. FIG. 19 (F) is a photograph,
Alkaline Phosphatase (ALP) stained after ten days culture, showing a bone
filling material on which osteoblast-like cell lines MC3T3 were cultured.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062]Hereinafter, a method for producing a bone filling material of the
present invention is explained. The method for producing a bone filling
material of the present invention basically comprises the steps of: (a)
kneading ingredient comprising calcium-based material and material
comprising binder; (b) molding a molded body having a predetermined shape
from a kneaded material obtained in step (a) with an injection molding
machine having a mold; (c) removing the binder contained in the molded
body (i.e., degreasing) to obtain a degreased body, the molded body being
obtained in step (b); and (d) heating and sintering the degreased body to
obtain a sintered body, the degreased body being obtained in step (c).
The method may include publicly known steps such as an after treatment
step for a molded body.

[0063]Since each of the bone filling material obtained by the production
method of the present invention has uniformity in size, appropriate
dosage of pharmaceutical agent can be administered, even when the
pharmaceutical agent is incorporated in the bone filling material.
Furthermore, the bone filling materials, when administered, have
appropriate porosity while maintaining the strength of each bone filling
material, because the bone filling material has uniform density and its
size can be controllable. Each steps of the method for producing a bone
filling material is explained in the following.

Kneading Step

[0064]In the kneading step, ingredient comprising calcium-based material
and material comprising binder are kneaded. In this step, it is preferred
to use powdered ingredient. In this step, powdered ingredient and sub
materials such as binders are mixed so that they are made to be suitable
for injection molding.

Kinds of Ingredient Powder

[0065]Calcium-based materials, for example, are used as powdered
ingredients. Examples of the calcium-based materials include calcium
phosphate-based materials, calcium carbonate-based materials, calcium
lactate, and calcium gluconate. Among them, calcium phosphate-based
material or calcium carbonate-based material is preferred. Specific
examples of calcium phosphate-based materials as powdered ingredients
include one or more than one kind of hydroxyl apatite, carbonic acid
apatite, fluorapatite, chlorapatite, β-TCP, α-TCP, calcium
metaphosphate, tetracalcium phosphate, calcium hydrogen phosphate,
calcium dihydrogen phosphate, calcium pyrophosphate, the salts thereof,
and the solvates and dihydrates thereof. Among them, β-TCP or
hydroxyl apatite is preferred. Specific examples of calcium
carbonate-based materials include calcium carbonate and calcium hydrogen
carbonate. Among them, calcium carbonate is preferred. Note that, the
powdered ingredients are not specifically limited to these materials, and
well-known materials used as ingredients of the bone filling materials
can be used as appropriate.

Size of the Ingredient Powder

[0066]When the size of the ingredient powder is too small, many binders
are required for forming a mold, and the physical properties of the
resultant bone filling material is deteriorated. On the other hand, when
the size of the ingredient powder is too large, there are risks that the
ingredient powders get into gaps between a screw and a cylinder of a
molding machine, or sintering of ingredient powder does not proceed. In
the present invention, powder injection molding is basically performed,
but the ingredient powder used in the powder injection molding is not
always metal powder. As a result of an experiment, the grain size of the
ingredient powder is, for example, from 0.01 μm to 100 μm (both
inclusive, same below), and is preferably from 0.1 μm to 20 μm. In
a general powder metallurgy, for example, powders with the size of 100
μm are used. For example, in Japanese Patent Laid-Open No. 2004-97259,
hydroxyl apatite having a grain size of less than 150 μm is used
(paragraph [0025]). But in the present invention, it is preferred that
ingredient powder having relatively small grain size be used to improve
fluidity of kneaded material (mixture of ingredient powders and binders)
and improve the density of a sintered body. On the other hand, although
the bone filling material of the present invention requires certain
strength, it is assumed to be eroded by osteoclasts. From this
perspective, the grain size may be from 0.1 μm to 50 μm, and may
preferably be from 0.5 μm to 10 μm.

Sub Materials

[0067]In the kneading step, materials such as binder, other than the
ingredient, are mixed with the ingredient. The examples of the binder
include (meta) acrylic-based resin, wax lubricant, (preferably,
thermoplastic resin other than (meta) acrylic-based resin) and material
having lubricant. The example of methacrylic-based resin or acrylic-based
resin is methacrylate resin or acrylate resin, and it specifically
includes a polymer of n-butyl methacrylate or methyl methacrylate, or a
copolymer of n-butyl methacrylate and methyl methacrylate. The molecular
weight of methacrylic-based resin or the acrylic-based resin is not
specifically limited, but it is preferred to be adjusted as appropriate
so that the physical properties of the resultant bone filling material
are not lost, and the weight average molecular weight is, for example,
1×103 to 1×105. The content of methacrylic-based
resin or acrylic-based resin in the binders is not specifically limited,
but it is preferred to be adjusted as appropriate so that the physical
properties of the resultant bone filling material are not lost, and the
content is, for example, 1% by weight to 50% by weight.

[0068]The example of the wax lubricant is wax having a melting point of
40° C. to 100° C., and the melting point is preferably
40° C. to 70° C. As the wax having the above melting point,
for example, well known paraffin wax can be used as appropriate. A molded
body can be easily removed from a mold at the time of injection molding
by using wax having the above melting point. It is more preferred to use
wax having a melting point of 60° C. to 65° C., because a
molded body can be removed from a mold without cooling the mold too much.

[0070]The molecular weight of wax lubricant is not specifically limited,
but it is preferred to be adjusted as appropriate so that the physical
properties of the resultant bone filling material are not lost, and the
weight average molecular weight is, for example, 1×102 to
1×106. The content of wax lubricant in the binders is not
specifically limited, but it is preferred to be adjusted as appropriate
so that the physical properties of the resultant bone filling material
are not lost, and the content is, for example, 1% by weight to 50% by
weight.

[0071]The examples of a thermoplastic resin include one or more than one
kind of: polyacetal resin, (meta) acryl resin, polyolefin resin,
ethylene-vinyl acetate copolymer, and polyvinyl butyral. However, in the
present invention, resins other than (meta) acrylate resin and (meta)
acrylic-based resin are preferred as thermoplastic resins. Among them,
ethylene-vinyl acetate copolymer is preferred.

[0072]The molecular weight of a thermoplastic resin is not specifically
limited, but it is preferred to be adjusted as appropriate so that the
physical properties of the resultant bone filling material are not lost,
and the weight average molecular weight is, for example, 1×103
to 1×105. The content of a thermoplastic resin in the binders
is not specifically limited, but it is preferred to be adjusted as
appropriate so that the physical properties of the resultant bone filling
material are not lost, and the content is, for example, 1% by weight to
50% by weight.

[0073]The examples of lubricant (other than wax lubricant) include one or
more than one kind of: stearic acid, a salt of stearic acid, a hydrate of
stearic acid, a hydrate of a salt of stearic acid, and C1-C5
alkyl stearic acid (C1-C5 alkyl represents an alkyl group
having 1 to 5 carbon atoms, same below); or one of these materials and
polyethylene glycol or one of these materials and polyglycerol. The
content of lubricant in the binders is not specifically limited, but it
is preferred to be adjusted as appropriate so that the physical
properties of the resultant bone filling material are not lost, and the
content is, for example, 0.5% by weight to 15% by weight. The molded
bodies can be removed from a mold easily by using the above lubricant.
The lubricant may also act as a dispersing agent.

[0074]The phthalic acid ester group is another compound comprising the
binders. It is reported that the phthalic acid ester group is harmful to
the human body. But in a preferred embodiment of the present invention,
the binders are thermally decomposed almost completely. So chemical
compounds having poor biocompatibility, such as the phthalic acid ester
group, can be contained in binders. The example of the phthalic acid
ester group is C1-C5 alkyl phthalate such as dibutyl phthalate.
As demonstrated in the example described below, a bone filling material
having more preferred physical properties could be obtained by
consciously using the phthalic acid ester group.

[0075]The molecular weight of the phthalic acid ester group is not
specifically limited, but it is preferred to be adjusted as appropriate
so that the physical properties of the resultant bone filling material
are not lost, and the weight average molecular weight is, for example,
1×104 to 1×107. The phthalic acid ester group with
poor volatility is also preferred. The content of the phthalic acid ester
group in the binders is not specifically limited, but it is preferred to
be adjusted as appropriate so that the physical properties of the
resultant bone filling material are not lost, and the content is, for
example, 0% by weight to 20% by weight, and is preferably 0.5% by weight
to 15% by weight.

Binder Loadings

[0076]Binders are removed by being thermally decomposed in the binder
removing step described below. The parts where binders existed basically
become voids. So the porosity and the intensity of a resultant bone
filling material can be adjusted by controlling the quantity of binders
added to ingredient. But in general, the amount of binders required is
the amount that is enough to fill spaces between particles of
ingredients. This is because if the amount of binders is not enough,
appropriate fluidity can not be obtained, which in turn causes injection
molding defects such as short mold and weld, and also causes variations
in the shape or the density of the resultant molded bodies. Binder
Loadings are, for example, between 10 percent by weight to 100 percent by
weight, based on 100 percent by weight of ingredients, or may be 20
percent by weight to 50 percent by weight. The blending ratio of binders
to ingredients is 25 to 70 volume percent, preferably 30 to 55 volume
percent, and more preferably 35 to 45 volume percent.

Addition of Glass Component

[0077]A preferred embodiment of the present invention is a method for
producing a bone filling material comprising "ingredient comprising
calcium-based material, and material comprising binder", and glass
component. As the glass component, the following materials can be used as
appropriate: silica glass which is composed mostly of silicon dioxide;
borosilicate glass containing 5 to 20% by weight of B2O3; lead
glass containing 5 to 40% by weight of lead; potassium glass containing 5
to 30% by weight of potassium; Fluoroaluminosilicate glass including
sodium fluoride, aluminum fluoride, and the strontium fluoride; or a
mixture of one of these glasses and one kind or a mixture of more than
one kind of boric acid, lanthanum oxide, gadolinium oxide, niobium oxide,
zirconium oxide, and barium. The sinterability of a sintered body is
lowered by consciously adding glass components. As a result, minute
cracks and porosities are formed on the surface or inside the sintered
body, thereby producing a bon filling material which is suitable for
culturing cells. As glass component, the following materials or an
appropriate mixture thereof may be used: titanium, titanium alloy,
cobalt-chromium alloy, stainless steel, alumina, zirconia. Calcium
phosphate-based crystals such as apatite (Ca10(PO4)6O), or
calcium phosphate-based crystals such as
CaO--SiO2--MgO--P2O5 based crystallized glass may also be
used.

[0078]It is preferred that the glass component loadings are adjusted as
appropriate based on the physical properties required for the bone
filling material, but the loadings are, for example, between 1 percent by
weight to 20 percent by weight, based on 100 percent by weight of
ingredients, or may be 2 percent by weight to 10 percent by weight. The
blending ratio of glass components to ingredients is 1 to 20 volume
percent, preferably 2 to 10 volume percent, more preferably 3 to 10
volume percent.

Addition of Salt or Sugar

[0079]A preferred embodiment of the present invention is a method for
producing a bone filling material comprising "ingredient comprising
calcium-based material, and material comprising binder", and salt or
sugar (preferably salt). The sinterability of a sintered body is lowered
by consciously adding salt or sugar. As a result, minute cracks and
porosities are formed on the surface or inside the sintered body, thereby
producing a bone filling material which is suitable for cell culture.
And, by immersing the obtained bone filling material in water, salt or
sugar can be removed, thereby producing a bon filling material which is
suitable for cell culture. Well-known salts or sugars can be used as
appropriate. Salts which can be dissolved in water but is not thermally
decomposed at temperature that thermally decomposes binders, particularly
inorganic salt, is preferred. Specific examples of the salts include
sodium chloride, potassium chloride, calcium chloride or calcium
carbonate. Well-known sugars such as sucrose, glucose, and fructose can
be used as appropriate. Meanwhile, a preferred embodiment of the present
invention comprises thermally-degradable components with or without sugar
or salt. The word thermally-degradable component represents a component
which is not thermally-degraded in the kneading step, but is
thermally-degraded in the molding step and the sintering step, or is
thermally-degraded at a higher temperature than the heating temperature
of the molding step or the sintering step. Since the thermally-degradable
components are thermally-degraded somewhere in the molding step, the
sintering step, or after the sintering step, a bone filling material
having appropriate voids can be obtained.

[0080]It is preferred that salt or sugar loadings are adjusted as
appropriate based on the physical properties required for the bone
filling material, but the loadings are, for example, between 1 percent by
weight to 20 percent by weight, based on 100 percent by weight of
ingredients, or may be 2 percent by weight to 10 percent by weight. The
blending ratio of salt or sugar to ingredients is 1 to 30 volume percent,
preferably 2 to 20 volume percent, more preferably 3 to 10 volume
percent. When thermally-degradable components are added to ingredients,
the same amount as salt or sugar is preferred to be added.

Kneading

[0081]In the kneading step, compound which is a material for injection
molding is obtained by mixing the above described ingredient powders and
binders. If ingredient powders are not uniformly mixed, several problems
will be caused. For example, the geometry of a molded body will be
deteriorated. In particular, it is preferred that each bone filling
material of the present invention have constant geometry in order to
maintain the same dosage of pharmaceutical agents. So it is desirable
that the ingredients be made as uniform as possible.

[0082]The temperature of the kneading step is preferred to be adjusted as
appropriate based on the kind of binders. But if the temperature is low,
the ingredient powders and the binders can not be mixed, and if the
temperature is high, the binders will be thermally decomposed. So the
temperature is set to be, for example, from 110° C. to 240°
C., preferably from 130° C. to 190° C., and more preferably
from 140° C. to 160° C.

[0083]In the kneading step, it takes long time for kneading ingredients
uniformly. But if the kneading time is too long, the binders will be
thermally decomposed. So the kneading time is preferred to be adjusted as
appropriate based on the kinds on binders. The kneading time is, for
example, from 30 minutes to 5 hours. It may also be 45 minutes to 1.5
hours.

[0084]As a kneading machine used in the kneading step, for example, a
pressure type kneader, or a uniaxial or biaxial extrusion kneader can be
used as appropriate. Since the bone filling material of the present
invention is pharmaceutical agent which will be used for transplant, it
is preferred that the bone filling material be free of impurities such as
broken pieces of kneading blades of a kneading machine. From this
perspective, it is desired that the kneading blades be made of material
with high hardness, and kneading blades which are provided by surface
protective layers (e.g., deposited by TiN coating) are preferred.

[0085]A preferred kneading process is, for example, as follows. Firstly, a
kneading machine is heated to a predetermined temperature, and binder
having high melting point is cast into the kneading machine. When the
binder is dissolved to a certain extent, ingredient powders are cast into
the kneading machine. After that, binder having low melting point and
ingredient powders are cast into the kneading machine, and 1/2 to 4/5 by
volume of ingredient is cast into the kneading machine, followed by
casting low-volatile components such as DBP (dibutyl phthalate). And
then, the remaining ingredient is cast into the kneading machine. In this
way, the aggregation of the ingredient powders could be dispersed by
kneading binder having high melting point (high-viscous binder) and
ingredient powders in the beginning of the step.

[0086]In particular, for example, the kneading step comprises the steps
of: putting the (meta) acrylic-based resin and the ethylene-vinyl acetate
copolymer in a kneading machine; putting the ingredient, the paraffin
wax, and the stearic acid in the kneading machine while kneading the
(meta) acrylic-based resin and the ethylene-vinyl acetate copolymer; and
putting the dibutyl phthalate in the kneading machine while kneading the
(meta) acrylic-based resin, the ethylene-vinyl acetate copolymer, the
paraffin wax, and the stearic acid. In this way, the compound which is
material for injection molding can be obtained.

[0087]However, since the bone filling material of the present invention is
replaced with bones in the future, molded products having consciously
made minute cracks may be used to promote the replacement. From this
perspective, for example, the kneading time may be from 15 minutes to 30
minutes, and the kneading temperature may be from 80° C. to
100° C.

Molding Step

[0088]The molding step is a process for producing a molded body with a
predetermined shape by injection molding. It is preferred that a bone
filling agent have four protruding parts extending from the center of the
regular tetrahedron form of the bone filling material toward each vertex
thereof. Herein after an example of a mold for producing the bone filling
material is explained. FIG. 1 is a conceptual diagram showing an example
of a mold used in the method for producing a bone filling material of the
present invention. Reference number 1 of FIG. 1 represents the shape of
the mold with its grooves get together. Reference number 2 represents a
part corresponding to an injection hole. Reference number 3 represents a
parting face. And reference number 4 represents a wedge. The above
described mold, for example, has: a fixed mold having an inlet (gate)
where material is injected; and a movable mold which is contacted with
the fixed mold when the material is injected, and is apart from the fixed
mold after a molded product is formed. And the inlet of the mold is
located at a tip part of one of the protruding parts so that the material
for injection molding is injected therefrom, and the parting face of the
fixed mold and the movable mold is located at the other three protruding
parts, wherein the movable mold has ejector pins located inside the other
three protruding parts. The term "inside the other three protruding
parts" means the direction from the bottom of the FIG. 1(A) to the
reference number 2.

[0089]The bone filling material above described has a plurality of
protruding parts (e.g., more than four protruding parts). And the mold
comprises: a fixed mold having an inlet where material is injected; a
movable mold being contacted with the fixed mold when the material is
injected, the movable mold being apart from the fixed mold after a molded
body is formed, wherein the inlet of the fixed mold is located at a tip
part of one of the protruding parts so that the material for injection
molding is injected from the tip part of the protruding part, wherein a
parting face of the fixed mold and the movable mold has an inclined
surface, the inclined surface inclines toward the inlet of the fixed mold
from the edge face to the center of the parting face, and wherein the
movable mold has dent parts for providing predetermined wedges to the
movable mold. As shown in FIG. 1, the parting faces provided at the parts
corresponding to the protruding parts are not horizontally arranged but
are inclined toward the inlet of the mold as it extends to the center of
the protruding parts.

[0090]Producing a bone filling material with one molding process
accompanies difficulties. Those who skilled in the art think of a method
for injection molding in which protruding parts independently produced
are combined together. But since the bone filling material of the present
invention is minute, it is difficult to produce the protruding parts
separately and combine them together. The mold of the present invention
can produce an injected body having a plurality of protruding parts in
one injection molding operation. The mold of the present invention also
made it easy for a molded body to be taken out of the fixed mold, for
example, by providing a three-dimensional parting face, wherein the
parting face of the fixed mold and the movable mold has an inclined
surface toward the inlet from the edge to the center of the parting face.
For example, when a split mold having a flat parting face is used to mold
a molded body, the molded body is highly likely to be stuck in a fixed
mold, and it is difficult to remove the molded body from the fixed mold.
But the above described mold having inclined parting face lowered the
possibility of a molded body remained in a fixed mold.

[0091]A preferred embodiment of a mold of the present invention has wedges
(4) with the height of 1 μm to 1×102 μm (preferably 5
μm to 2×10 μm, or 5 μm to 1×10 μm). The shape of
the wedge is not specifically limited, if it served as a wedge, and
well-known shape of wedge can be adopted. In FIG. 1, three wedges are
provided at the parts corresponding to the tip of each protruding part.
But the number or the position of the wedges is not particularly limited,
and the wedges may be provided at the parts other than the tips of the
protruding parts (e.g., the body part of the protruding part). If a
molded body remains in the fixed mold, bone filling materials can not be
produced. The wedge part serves as a wedge to the moving part, which
prevents a molded body being left in the fixed mold when the mold is
opened.

[0092]A preferred embodiment of the present invention is one of the above
described methods for producing a bone filling material, wherein the
movable mold has one or a plurality of grooves (preferably parallel
grooves or spiral-shaped grooves) with the depth of 1 μm to 2×10
μm (preferably 5 μm to 1×10 μm). If a molded body remains
in the fixed mold, bone filling materials can not be produced. The groove
parts increase the surface area of the movable mold part, thereby
preventing a molded body from being left in the fixed mold when the mold
is opened. The groove part is provided, for example, on the body part of
the protruding parts.

[0093]A preferred embodiment of the present invention is one of the above
described methods for producing a bone filling material, wherein the bone
filling material has four protruding parts and the movable mold has
ejector pins located inside the protruding parts. It is very important
for injection molding to remove a molded body from a mold. In this
embodiment, a protruding pin is arranged so that it is located inside a
protruding part (so that the pin is directed from the other end of the
tip of a protruding part through the center of the mold toward the tip of
the protruding part). So a molded body can be taken out effectively.

[0094]A preferred embodiment of the present invention is one of the above
described methods for producing a bone filling material, wherein the bone
filling material has a plurality of protruding parts. And the mold
comprises: a fixed mold; a movable mold being contacted with the fixed
mold when material is injected, the movable mold being apart from the
fixed mold after a molded body is formed, wherein an inlet of the
material for injection molding is located at a parting face of the fixed
mold and the movable mold, wherein the parting face of the fixed mold and
the movable mold has an inclined surface, the inclined surface inclines
toward the center of the bone filling material from the edge face to the
center of the parting face, and wherein the movable mold has groove parts
for providing predetermined wedges to the movable mold. A molded body
having a plurality of protruding parts can be obtained in one injection
molding operation by an ingenious mold at the timing of injection
molding.

[0095]In the molding step, an injection molding is performed preferably by
using an injection molding machine. The injection molding machine is not
specifically limited, and a well-known injection molding machine can be
used. The examples of the injection molding machine include: a vertical
injection molding machine or a horizontal injection molding machine; a
high pressure injection molding machine, a moderate pressure injection
molding machine, or a low pressure injection molding machine; or a
plunger injection molding machine or a screw injection molding machine.
However, in order to produce a bone filling material having minute
protruding parts from calcium phosphate-based material, an injection
molding machine which is a horizontal screw type injection molding
machine (which is preferably a high pressure injection molding machine)
can be preferably used. However, if impurities such as broken pieces of a
screw cylinder are mixed in the bone filling material of the present
invention, (although it will not be a problem for an ordinary molded
body) it will be a problem because the bone filling material is intended
to be administered in vivo. So it is preferred that surface protective
layers such as TiN coating layers are preferred to be formed on the
surface of the screw.

Binder Removal Step

[0096]In the binder removal step, binder contained in the molded body
obtained in the above described molded step is removed, and thereby
degreased body is produced. The binder removal step is also referred to
as a degreasing step. If binder is not removed sufficiently in the binder
removal step, the molded body may be cracked or bloated in the sintering
step below. In the degreasing step, it is expected that the binder
removal is completed without causing defects on the molded body such as
deformations and cracks. The examples of binder removal method include
the sublimation method, the natural drying method, the solvent extraction
method, the thermal degreasing method, and the like, and the thermal
degreasing method is preferred. The thermal degreasing method is
performed in an ambient atmosphere, a reduced pressure atmosphere, a
pressurized atmosphere, a gas atmosphere, or the like, and is preferably
performed in an ambient atmosphere. A molded body is preferably placed on
a ceramics setter (porous and dense) when it is cast into a degreasing
furnace. If the molded body is large (i.e., thick molded body), porous
setter such as alumina setter is preferred. It is also desirable to watch
for impurities of contaminated setter and components of a heated setter.

[0097]A binder removal step, for example, has several stages of heating-up
period and duration period in accordance with the pyrolysis temperature
of resin contained in binder. In particular, effective pyrolysis of resin
having low pyrolysis temperature improves sintering performance. In the
present invention, since temperature is raised as above described, resin
having low pyrolysis temperature can be effectively pyrolyzed. A
preferred embodiment of a bone filling material of the present invention
may include compounds having poor biocompatibility in binder, although
the bone filling material is administered in vivo. Such compound tends to
be a binder having low melting point. So in the heating-up step, in order
to vaporize binder having low melting point completely, it is preferred
that the temperature be raised relatively moderately. A specific example
of heating up ratio is at 1° C./hour to 3×102°
C./hour until the temperature reaches in the range of 110° C. to
300° C. which is the temperature of the first maintaining period
(preferably until the temperature reaches in the range of 230° C.
to 250° C.), preferably at 1×10° C./hour to
2×102° C./hour, more preferably at 2×10°
C./hour to 5×10° C./hour, and the ratio may also be at
3×10° C./hour to 4×10° C./hour. The maintaining
step is, for example, from 2×10 minutes to 5 hours, preferably from
3×10 minutes to 2 hours.

Sintering Step

[0098]The sintering step is a step for heating a molded body obtained in
the binder removal step. Japanese Patent Laid-Open No. 2004-97259
(paragraph [0025]) shows a sintering at 1,250° C. for an hour. But
in a preferred embodiment of the present invention, a molded body is
heated from ambient atmosphere to the highest temperature
9×102° C. to 1.1×103° C. This is, for
example, to turn α-TCP, as ingredient, effectively into β-TCP.
High temperature maintaining period is, for example, 5×10-1
hours to 3 hours. It is noted that in the sintering step, a heating-up
step (and maintaining step) is followed by a cooling step, wherein
well-known cooling methods are used as appropriate. The sintering period
including the cooling period is, for example, from 6 hours to 5×10
hours, preferably from 1×10 hours to 3×10 hours. The molding
temperature is, for example, from 1×102° C. to
1.5×102° C. And the mold temperature is, for example,
from 1×10° C. to 3×10° C.

Aftertreatment Step

[0099]The aftertreatment step is an optional step for aftertreatment of
the molded body obtained in the sintering step. Specific examples of the
aftertreatment step include patching holes caused by ejector pins, and
cleaning the molded body.

[0100]It is another preferred embodiment of the present invention to add
well-known pharmaceutical agent in addition to ingredient powder. The
bone filling material containing the pharmaceutical agent serves as
carriers thereof because the volume of the bone filling material produced
in the present invention is approximately uniform. It is preferred that
the pharmaceutical agent added to the bone filling material remain
activity at high temperatures.

Impregnation and Administration of Pharmaceutical Agent

[0101]Another preferred embodiment of the present invention is a bone
filling material (or a sintered body obtained in the sintering step) to
which a pharmaceutical agent is impregnated or administered as needed.
The methods for administering pharmaceutical agent includes immersion
administration, spray administration, and spin coat administration,
wherein pharmaceutical composition which is obtained by dissolving
pharmaceutical agent with well-known pharmaceutically acceptable diluent
(solvent) is administered. Among them, immersion administration is
preferred. Immersion administration of pharmaceutical agent impregnates
the surface or inside of the bone filling agent with pharmaceutical
agent. Namely, the present invention can provide a bone filling material
to which a predetermined pharmaceutical agent is impregnated or
administered.

[0102]A preferred embodiment of the bone filling material of the present
invention is the above described bone filling material wherein the
pharmaceutical agent comprises an osteogenesis/chondrogenesis promoter
(including chondrogenesis promoting factor), a joint disease therapeutic
agent, a preventive and/or therapeutic agent for bone/cartilage disease,
a bone-regenerating agent, a bone resorption-suppressing substance, an
angiogenesis promoter, an antibacterial agent, an antibiotics, or an
anticancer agent. A preferred embodiment of the bone filling material of
the present invention is the above described bone filling material
wherein the pharmaceutical agent comprises thienoindazole derivative
represented by the below described general formula (I). The
thienoindazole derivative (4,5-dihydro-1-methyl-1H-thieno[3,4-g] indazole
derivative) can be produced in accordance with a method disclosed in the
Japanese Patent Laid-Open No. 2002-356419. It is desirable that effective
dose of the pharmaceutical agent that provides a predetermined efficacy
is contained in the bone filling material of the present invention.
Namely, since well-known pharmaceutical agent can be used in, the present
invention, the amount of pharmaceutical agent contained in the bone
filling material is preferred to be adjusted as appropriate so that the
effective dose of the pharmaceutical agent that provides a predetermined
efficacy can be administered.

##STR00003##

[0103]wherein R1V represents a carboxyamido group
(--CH(NH2)(CO2H), --CH(NH2)(SO3H),
--CH(NH2)(SO2H), --CH(NH2)(SO2NHRII),
--CH(NH2)(PO(NH2)OH) and --CH(NH2)(PO(ORII)OH),
(where RII is a C1-5 linear alkyl group). Among them,
carboxyamido group is the most preferred.

Osteogenesis/Chondrogenesis Promoter

[0104]As the osteogenesis/chondrogenesis promoter, a publicly known agent
for inducing osteogenesis or chondrogenesis can be used as needed. In
particular, a chondrogenesis promoter is, for example, a
2-[1-(2,2-Diethoxy-ethyl)-3-(3-p-tolyl-ureido)-2,3-dihydro-1H-indol-3-yl]-
-N-p-tolyl-acetamide which is disclosed in WO 02/087620. As a
chondrogenesis promoter, for example, osteogenesis promoting factor can
be used. The osteogenesis promoting factor is generally referred to as
BMP (bone morphogenetic protein). The BMP is a substance for
bone/cartilage induction which acts on undifferentiated mesenchymal cells
from outside, differentiating them to chondrocyte or osteoblasts. As the
osteogenesis promoting factor, for example, BMP1 to 13 can be used. The
BMP used as a pharmaceutical agent of the present invention may be either
one of the BMP obtained by genetic recombination or the purified BMP
taken form Dunn osteogenic sarcoma (see Takaoka, K., Biomedical Research,
2 (5) 466-471 (1981)).

[0106]Examples of the bone/cartilage disease preventing or treating agent
include one or mixtures of more than one kind of the following
substances: non-peptide osteogenesis-promoting substances such as
prostaglandin A1 derivative, vitamin D derivative, vitamin K2
derivative, eicosapentaenoic acid derivative, benzylphosphonic acid,
bisphosphonic acid derivative, sex hormone derivative,
phenolsulfophthalein derivative, benzothiopyran or benzothiepine
derivative, thienoindazole derivative, menatetrenone derivative,
helioxanthine derivative; and a hardly soluble peptide
osteogenesis-promoting substance. These substances can be obtained by a
known method. A bone/cartilage disease preventing agent includes one or
both of a cartilage disease preventive agent and an agent preventing the
situation that a bone/cartilage disease develops.

Bone-Regenerating Agent

[0107]Examples of the bone-regenerating agent include one kind or a
mixture of more than one kind of the following substances: calmodulin;
actinomycin D; cyclosporin A; glucosamine sulfate; glucosamine
hydrochloride; marrow extract; calcium phosphate; lactic acid/glycolic
acid/ε-caprolactone copolymer; platelet rich plasma; or human
marrow mesenchymal cell. These substances can be obtained by a known
method.

Bone Resorption-Suppressing Substance

[0108]Examples of the bone resorption-suppressing substance include one
kind or a mixture of more than one kind of estrogenic agent, calcitonin,
and bisphosphonate. These substances can be obtained by a known method.

Angiogenesis Promoter

[0109]Examples of the angiogenesis promoter include one kind or a mixture
of more than one kind of the following substances: indigocarmine;
4-[N-methyl-N-(2-phenylethyl) amino]-1-(3,5-dimethyl-4-propionyl
aminobenzoyl) piperidine;
4-(5H-7,8,9,10-tetrahydro-5,7,7,10,10-pentamethyl
benzo[e]naphtho[2,3-b][1,4]diazepine-13-yl) benzoic acid; activated
protein C; urotensin II-like peptide compound; fibroblast growth factor
(FGF) (including basic FGF and acidic FGF); vascular endothelial cell
growth factors (VEGF) (preferably a platelet-derived factor); hepatocyte
growth factor (HGF); angiopoetin (including angiopoetin-1 and
angiopoetin-2); platelet-derived growth factor (PDGF), Insulin-like
growth factor (IGF) or smooth muscle embryo myosin heavy chain (SMemb).
Of these substances, fibroblast growth factor is preferred (see, Hockel,
M. et al., Arch. Surg., No. 128, p. 423, 1993). A basic fibroblast growth
factor (bFGF) is preferred as a fibroblast growth factor, and a specific
example includes trafermin (gene recombinant). Namely, a preferred
embodiment of the bone filling material of the present invention is the
above described bone filling material comprising trafermin, a salt
thereof, a solvate thereof, or a prodrug thereof. "A salt thereof"
represents a salt of trafermin, and is specifically the same salt as
above explained. "A solvate thereof" represents a solvate of trafermin,
and is specifically the same solvate as above explained. "A pro drug
thereof" represents a prodrug of trafermin, and represents an agent that
turns into, for example, trafermin, an ion thereof, or a salt thereof in
vivo after administration. In particular, a prodrug contains protecting
groups such as an amino group which are taken off in vivo, and acts the
same as trafermin.

[0111]Anticancer agent is a pharmaceutical agent for treating or
preventing cancer. A publicly known anticancer agent can be used as
needed. Specific examples of anticancer agent include the following
substances: anticancer hemolytic streptococcus formulation such as OK-432
(commercial name Picibanil); anticancer polysaccharide such as krestin,
lentinan, schizophyllan, and sonifilan; anticancer antibiotics such as
mitomycin C (commercial name Mitomycin, etc.), actinomycin D (commercial
name Cosmegen), bleomycin hydrochloride (commercial name Bleo), bleomycin
sulfate (commercial name Bleo S), daunorubicin hydrochloride (commercial
name Daunomycin), doxorubicin hydrochloride (commercial name Adriacin),
neocarzinostatin (commercial name Neocarzinostatin), aclarubicin
hydrochloride (commercial name Aclacinon), or epirubicin hydrochloride
(commercial name Farmorubicin); mitotic inhibitor such as Vinblastine;
alkylating agent such as cis-platin, carboplatin, and cyclophosphamide;
antimetabolite such as 5-fluorouracil, cytosine arabinoside and
hydroxyurea,
N-{5-[N-(3,4-dihydro-2-methyl-4-oxoquinazoline-6-ylmethyl)-N-methylamino]-
-2-thenoyl}-L-glutamic acid; anticancer antibiotics such as adriamycin and
bleomycin; enzyme such as asparaginase; topoisomerase inhibitor such as
etoposide; biological response modifier such as Interferon; antiestrogen
such as "NOLVADEX" (tamoxifen); antiandrogen such as "CASODEX";
antimetabolite such as Fluorouracil, Tegafur, Tegafur-uracil, and
Methotrexate; plant alkaloid such as Vncristine; anticancer antibiotic
such as mitomycin C, actinomycin D, bleomycin hydrochloride, bleomycin
sulfate, daunorubicin hydrochloride, doxorubicin hydrochloride,
neocarzinostatin, aclarubicin hydrochloride, Aclacinon, and epirubicin
hydrochloride; and platinum complex such as cyclotriphosphazene-platinum
complex, and cisplatin-platinum complex.

[0112]It is expected that the bone filling material of the present
invention promotes its replacement with bone tissues in vivo, so
pharmaceutical agents including a specific polypeptide or gene may be
administered or impregnated to the bone filling material. The examples of
the polypeptide or the gene include a basic fibroblast growth factor
(bFGF), a platelet derived growth factor (PDGF), insulin, an insulin-like
growth factor (IGF), a hepatocyte growth factor (HGF), a glial cell
line-derived neurotrophic factor (GDNF), a neurotrophic factor (NF),
hormone, cytokine, a bone morphogenetic factor (BMP), a transforming
growth factor (TGF), a vascular endothelial cell growth factor (VEGF).
Among them, a growth factor promoting neoangiogenesis and/or osteogenesis
is preferred. The examples of the growth factor include a bone
morphogenetic factor (BMP), a bone growth factor (BGF), a vascular
endothelial cell growth factor (VEGF) and a transforming growth factor
(TGF). The specific example is a calponin gene disclosed in the Japanese
Patent No, 3713290. The gene is preferred to be contained in the bone
filling material as much as the amount which is effective for the gene
therapy. It is also preferred that the gene be contained in the bone
filling material as it is (naked), in a micelle state, or in the form of
a recombinant vector which is transformed into a known vector such as a
virus vector. The pharmaceutical agent may be a known genetic antibody.

[0113]The gene can be adjusted based on well-known base sequence in
accordance with ordinary methods. For example, cDNA of the targeted gene
is adjusted in the following method. RNA is extracted from osteoblasts,
and primer is produced based on a well-known base sequence, and then
cloning by PCR method. Also commercially available genes may be used.

[0114]A preferred embodiment of a bone filling material of the present
invention is the above described bone filling material including
stabilizer. As the stabilizer, a well-known stabilizer used for polymer
compound, in particular, pharmaceutically acceptable stabilizing agents
can be used as appropriate. The strength of the bone filling material of
the present invention is maintained mainly in vivo for extended period.
But it is assumed that the bone filling material is decomposed in the
early stages due to the existence of enzymes such as protease. So a
preferred embodiment of the present invention includes inhibitors such as
protease inhibitor. Well-known enzyme inhibitor can be used for the
inhibitor as appropriate. The specific examples include one or more than
one kind of the following substances: 4-(2-aminoethyl) benzene sulfonyl
fluoride, aprotinin, bestain, calpains inhibitor I, calpains inhibitor
II, chymostain, 3,4-Dichloroisocoumain, E-64, EDTA, EGTA, lactacystin,
leupeptin, MG-115, MG-132, pepstain A, phenylmethyl sulfonyl fluoride,
proteasome inhibitor I, p-toluene sulfonyl-L-lysine chloromethylketone,
p-toluene sulfonyl-L-phenylalanine chloromethylketone, or tyrosine
inhibitor. These protease inhibitors are commercially available, and the
inhibitory concentrations thereof are also commonly known. A preferred
embodiment of the compound formed by the bone filling material of the
present invention maintains the strength for a prolonged period and has
sustained drug release. Therefore, the bone filling material of the
present invention preferably contains 2 to 100 times of one dosage of the
above protease inhibitor, more preferably contains 2 to 50 times thereof.
The specific dose level of the protease inhibitor differs based on the
kind of the protease inhibitor to be used. The dose preferably contains
the amount of protease inhibitor that makes inhibitor's function
effective (i.e., the effective dose). In general, the bone filling
material (per 1 g) contains 0.1 μg to 0.5 μg of protease inhibitor.
The amount included may be 1 μg to 0.1 mg, or may be 10 μg to 0.1
mg. The specific amount of dosage increases in almost proportion to the
volume of the site to which the bone filling material is administered.

Adhesiveness-Imparting Agent

[0115]Another preferred embodiment of the present invention is a bone
filling material (or, a sintered body obtained in the sintering process)
to which adhesiveness-imparting agents are impregnated or administered as
appropriate. When a heat-resistant adhesiveness-imparting agent is used,
the adhesiveness-imparting agent may be mixed with ingredient powders so
that a bone filling material which is powder blended with the
adhesiveness-imparting agent can be obtained (in this case, the
adhesiveness-imparting agent exists on the surface of the bone filling
material, and when the adhesiveness-imparting agent on the surface is
replaced with bone tissues, a new surface having the other
adhesiveness-imparting agent appears, thereby maintaining the
adhesiveness of the bone filling material). Also, a powdered
adhesiveness-imparting agent may be sprayed on the surface of a molded
body or a sintered body. Furthermore, adhesiveness-imparting agents may
be added to the surface of the bone filling material by powder blending,
wherein a plurality of bone filling agents and powdered
adhesiveness-imparting agents are mixed together and then agitated as
needed. The adhesiveness-imparting agent may be impregnated or
administered with the above mentioned pharmaceutical agent, and the
adhesiveness-imparting agent alone may be impregnated or administered.
The adhesiveness-imparting agent is an agent for raising adhesion
property between the bone filling materials, and it is preferred that the
adhesiveness-imparting agent alone do not have high adhesiveness but
increase adhesiveness when it contacts with cells in vivo. A specific
example of the adhesiveness-imparting agent is Thrombin. Thrombin is one
of enzymes which promotes blood clot. Thrombin produces fibrin which is a
blood clotting substance in vivo. Fibrin produced by thrombin promotes
blood clotting. So when thrombin is used as an adhesiveness-imparting
agent, the adhesiveness of the bone filling material will be improved,
which raises the strength of the bone filling material by fixing the bone
filling materials each other firmly. Thrombin can be impregnated or
administered with the same amount of the above described pharmaceutical
agent and in the same manner thereof.

[0116]A preferred embodiment of the present invention comprises the step
of: preparing a composition including two kinds of adhesiveness-imparting
agents; obtaining a first bone filling material group by impregnating a
certain bone filling material group with a first composition or by
administering a first composition to a certain bone filling material
group; obtaining a second bone filling material group by impregnating a
certain bone filling material group with a second composition or by
administering a second composition to a certain bone filling material
group, wherein the bone filling material groups including the first bone
filling material group and the second bone filling material group are
used as the bone filling materials. And for example, the first
composition comprises: one or more than one kind of compound represented
by the below-described general formula (I) or (II), or a first compound
including 3 to 8 repeating units represented by the below-described
general formula (III); and a first diluent (or a carrier), and the second
composition comprises: one or more than one kind of compound represented
by the below-described general formula (I) or (II), or a second compound
including 3 to 8 repeating units represented by the below-described
general formula (III); and a second diluent.

X1--(OCH2CH2)n--X2 (I)

[0117]wherein X1 and X2 are the same or different and each
represents --R1COONHS (where R1 is a C1-7 alkylene group),
--COR1COONHS (where R1 is a C1-7 alkylene group),
--NOCOR1--R2 (where R1 is a C1-7 alkylene group, and
R2 is a maleimide group), --R1NH2 (where R1 is a
C1-7 alkylene group), --R1SH (where R1 is a C1-7
alkylene group), or --CO2PhNO2 (where Ph is an o-, m-, or
p-phenylene group), and n is an integer of 80 to 1000.

##STR00004##

[0118]wherein XII-1 to XII-4 are the same or different and each
represents --R1COONHS (where R1 is a C1-7 alkylene group),
--COR1COONHS (where R1 is a C1-7 alkylene group),
--NOCOR1--R2 (where R1 is a C1-7 alkylene group, and
R2 is a maleimide group), --R1NH2 (where R1 is a
C1-7 alkylene group), --R1SH (where R1 is a C1-7
alkylene group), or --CO2PhNO2 (where Ph is an o-, m-, or
p-phenylene group), and to nII-1 to nII-4 are the same or
different and each represents an integer of 20 to 250.

##STR00005##

[0119]wherein XIII represents --R1COONHS (where R1 is a
C1-7 alkylene group), --COR1COONHS, --NOCOR1--R2
(where R1 is a C1-7 alkylene group, and R2 is a maleimide
group), --R1NH2 (where R1 is a C1-7 alkylene group),
--R1SH (where R1 is a C1-7 alkylene group), or
--CO2PhNO2 (where Ph is an o-, m-, or p-phenylene group), and
nIII represents an integer of 10 to 150.

[0120]In the general formula (I), X1 and X2 are the same or
different and each represents --R1COONHS, --COR1COONHS,
--NOCOR1--R2 (where R2 is a maleimide group),
--R1NH2, --R1SH, or --CO2PhNO2, and n is an
integer of 80 to 1000. In the general formula (I), X1 and X2
preferably are the same. R1 is a C1-7 alkylene group,
preferably is a C1-5 alkylene group, and more preferably is a
C1-2 alkylene group or a C5 alkylene group. Ph is an o-, m-, or
p-phenylene group, and preferably is a p-phenylene group. And, n is an
integer of 80 to 1000, and preferably is 100 to 500.

[0121]In the general formula (II), XII-1 to XII-4 are the same
or different and each represents --R1COONHS, --COR1COONHS,
--NOCOR1--R2 (where R2 is a maleimide group),
--R1NH2, --R1SH, or --CO2PhNO2, and nII-1
to nII-4 are the same or different and each represents an integer of
20 to 250. In the general formula (II), XII-1 to XII-4
preferably are the same. R1 is a C1-7 alkylene group,
preferably is a C1-5 alkylene group, and more preferably is a
C1-2 alkylene group or a C5 alkylene group. Ph is an o-, m-, or
p-phenylene group, and preferably is a p-phenylene group. And nII-1
to nII-4 preferably are the same and each represents an integer of
20 to 250, and preferably is an integer of 40 to 200.

[0122]In the general formula (III), XIII represents --R1COONHS,
--COR1COONHS, --NOCOR1--R2 (where R2 is a maleimide
group), --R1NH2, --R1SH, or --CO2PhNO2, and
nIII represents an integer of 10 to 150. In the general formula
(III), R1 is a C1-7 alkylene group, preferably is a C1-5
alkylene group, and more preferably is a C1-2 alkylene group or a
C5 alkylene group. Ph is an o-, m-, or p-phenylene group, and
preferably is a p-phenylene group. The number of repeating units of the
compound represented by the general formula (III) preferably is 3 to 5,
more preferably is 4. And the ends of the compound represented by the
general formula (III) preferably have functional groups represented by
XIII of the general formula (III). In particular, it is preferred
that the both ends of the compound have a group represented by a formula
XIII--(OCH2CH2)nIII--O--CH2--CH(--O--(CH2CH-
2O)nIII--XIII--CH2-- and a group represented by a
formula XIII--(OCH2CH2)nIII--O--CH2--CH(--O--(CH-
2CH2O)nIII--XIII) --CH2--O--.

[0123]A preferred embodiment of the adhesiveness-imparting agent of the
present invention is the above described adhesiveness-imparting agent,
wherein the first compound comprises one or more than one kind of
compounds represented by the general formula (I) or (II), where X1,
X2 or XII-1 to XII-4 are the same or different and each
represents --NOCOR1--R2 or --R1NH2, and wherein the
second compound comprises one or more than one kind of compounds
represented by the general formula (I) or (II), where X1, X2 or
XII-1 to XII-4 are the same or different and each represents
--COR1COONHS, --R1SH, or --CO2PhNO2 (where Ph is an
o-, m-, or p-phenylene group).

[0124]The first compound mixed in the first composition preferably has a
functional group of --NOCOR1--R2 or --R1NH2 (in
particular, a functional group of --R1NH2). It is also
preferred that the second compound, mixed with the first compound,
undergoes rapid crosslinkage reaction and the resultant compound has
predetermined strength (resilience) and morphological stability. It is
also desirable that the compound, which is obtained by mixing the first
compound with the second compound, has a certain steric structure and so
keep releasing proper amount of a pharmaceutical agent contained therein.
From this perspective, it is preferred that the second compound mixed in
the second composition have a functional group of --COR1COONHS,
--R1SH, or --CO2PhNO2 (where Ph is an o-, m-, or
p-phenylene group) (in particular, a compound having a functional group
of --COR1COONHS). Also, both the first and the second compounds
preferably are represented by the general formula (II). In particular, a
combination of a propylamine group and a succinimidyl group is preferred
as a combination of the functional group of the first compound and that
of the second compound.

[0125]Another preferred embodiment of the adhesiveness-imparting agent of
the present invention comprises the first compound and the second
compound, wherein the first compound comprises one or more than one kind
of compound represented by the general formula (I) or (II), where
X1, X2 or XII-1 to XII-4 are the same or different
and each represents --NOCOR1--R2 (where R1 is a C1-5
alkylene group) or --R1NH2, (where R1 is a C1-5
alkylene group), and wherein the second compound comprises one or more
than one kind of compound represented by the general formula (I) or (II),
where X1, X2 or XII-1 to XII-4 are the same or
different and each represents --COR1COONHS, --R1SH (where
R1 is a C1-5 alkylene group), or --CO2PhNO2 (where Ph
is a p-phenylene group).

[0126]The molecular weight of a compound composing the compound is not
specifically limited, but a preferred compound can be obtained from a
compound of molecular weight (number average molecular weight)
3×103 to 4×104, and a more preferred compound can
be obtained from a compound of molecular weight 1×104 to
3×104.

[0127]The concentration of a compound is not specifically limited if the
concentration of the compound composing the compound in the first
composition or the second composition are in the range of 5 mM to 20 mM.
A particularly preferred gel can be obtained from the gelator with the
above concentration. In particular, it is assumed that a particularly
effective compound can be obtained when the concentration of the
component in each composition is from 6 mM to 30 mM.

[0128]The compound represented by the general formula (I) or (II), and the
compound including 3 to 8 repeating units represented by the general
formula (III) are commercially available and can be synthesized by a well
known method.

[0129]The compound represented by the general formula (I) or (II), and the
compound including 3 to 8 repeating units represented by the general
formula (III) may respectively be a salt thereof or a solvate thereof.

[0130]The term "a salt thereof" represents a salt of the above described
compounds, particularly represents pharmaceutically acceptable salts of
the above described compounds. The term "pharmaceutically acceptable" in
this specification means that something is not deleterious to the
recipient thereof. The polyphosphoric acid of the present invention can
be made to salt by in an ordinary method. The examples of the salt
includes: the alkaline metal salts such as sodium salt, potassium salt,
and lithium salt; the alkaline earth metal salts such as calcium salt,
and magnesium salt; the metal salts such as aluminum salt, iron salt,
zinc salt, copper salt, nickel salt, and cobalt salt; the inorganic salts
such as ammonium salt; and the organic amine salts such as t-octyl amine
salt, dibenzylamine salt, morpholine salt, glucosamine salt,
phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine
salt, guanidine salt, diethylamine salt, triethylamine salt,
dicyclohexylamine salt, N,N-dibenzylethylenediamine salt, chloroprocaine
salt, procaine salt, diethanolamine salt, N-benzyl-N-phenethylamine salt,
piperazine salt, tetramethylammonium salt, tris (hydroxymethyl)
aminomethane salt. Among these salts, as polyphosphoric acid salt,
alkaline metal salt is preferred, and sodium salt is more preferred. In
this specification, "a salt thereof" may include not only anhydrous salt
but also hydrate salt. These salts, for example, are ionized in vivo, and
act the same as the above described compounds.

[0131]The term "a solvate thereof" represents a solvate of the above
described compounds. The solvate herein includes a hydrate. The agent of
the present invention may absorb moisture, be attached with absorption
water, and be hydrated when it is left in the atmosphere or
recrystallized. The solvates thus obtained are also included in "a
solvate thereof". These solvates are ionized in vivo, and act the same as
the above described compounds.

[0132]A preferred embodiment of the adhesiveness-imparting agent of the
present invention comprises a well known pharmaceutically acceptable
diluent as a diluent. Specific examples of the diluent are solvents
including one kind or a mixture of more than one kind of water, phosphate
buffer solution, citrate buffer solution, phosphate buffered saline or
physiologic saline solution.

[0133]The acid level of the diluent (or solvent) is not specifically
limited, but it includes pH 3 to pH 11. In order to improve the strength
of the gel obtained to some extent, the acid level is preferably from pH
5 to pH 10, more preferably form pH 6 to pH 9, and further preferably
from pH 7 to pH 8. The molar concentration (M) of the diluent is not
specifically limited, but it includes 1 mM to 1 M. In order to improve
the strength of the gel obtained to some extent, the molar concentration
is preferably from 5 mM to 300 mM, more preferably form 10 mM to 200 mM,
and further preferably from 15 mM to 100 mM.

[0134]A preferred embodiment of the adhesiveness-imparting agent of the
present invention is the above described adhesiveness-imparting agent
wherein one or both of the first composition and the second composition
include a stabilizing agent. As the stabilizing agent, a publicly known
stabilizing agent used for a polymer etc., in particular, a
pharmaceutically acceptable stabilizing agent can be used as needed. The
compound obtained from the adhesiveness-imparting agent of the present
invention is expected to maintain its strength for a prolonged period
mainly in vivo. Since enzymes such as protease exists in vivo, the
compound may be dissolved by the enzymes. So a preferred embodiment of
the bone filling material of the present invention includes inhibitors
such as protease inhibitor. As the inhibitors, publicly known enzyme
inhibitors can be used as needed. Specific examples of the protease
inhibitor include one or more than one kind of 4-(2-aminoethyl) benzene
sulfonyl fluoride, aprotinin, bestain, calpains inhibitor I, calpains
inhibitor II, chymostain, 3,4-dichloroisocoumain, E-64, EDTA, EGTA,
Lactacystin, Leupeptin, MG-115, MG-132, pepstain A, phenylmethyl sulfonyl
fluoride, proteasome inhibitor I, p-toluene sulfonyl-L-lysine
chloromethylketone, p-toluene sulfonyl-L-phenylalanine
chloromethylketone, or tyrosine inhibitor. These protease inhibitors are
commercially available, and the inhibitory concentrations thereof are
also publicly known. A preferred embodiment of a compound formed by the
adhesiveness-imparting agent of the present invention maintains the
strength for a prolonged period and has sustained drug release. So the
adhesiveness-imparting agent of the present invention preferably contains
2 to 100 times of one dosage of the above protease inhibitor, more
preferably contains 2 to 50 times thereof. The specific dose level of the
protease inhibitor differs based on the kind of the protease inhibitor to
be used. The dose preferably contains the amount of protease inhibitor
that makes inhibitor's function effective (i.e., the effective dose). In
general, the adhesiveness-imparting agent (1 mL) contains 0.1 μg to
0.5 mg of protease inhibitor. The amount included may be 1 μg to 0.1
mg, or may be 10 μg to 0.1 mg.

[0135]When the above described adhesiveness-imparting agent and a
pharmaceutical agent is used to form a compound, the resultant compound
comprising the adhesiveness-imparting agent contains the pharmaceutical
agent, thereby also acting as the bone filling material having sustained
drug release. The degradability of the compound can be controlled by
adjusting the structure of the adhesiveness-imparting agent and the
amount of stabilizing agent added, which makes it possible to adjust the
sustained release of the pharmaceutical agent. Namely, the present
invention can also provide a method for controlling release property of a
pharmaceutical agent by adjusting the amount of the stabilizing agent
added to the adhesiveness-imparting agent.

[0136]A preferred embodiment of the present invention is provided with a
covering layer on the surface of the bone filling agent. The thickness of
the covering layer is preferred to be adjusted as needed, but it is for
example 1 μm to 5×10 μm. And the covering layer includes
biocompatible compounds having hydrophilic groups. Examples of
hydrophilic group include an OH group, a COOH group, an NH3 group, a
CO3 group, and a SO3 group. It is preferred that the covering
layer include compounds having an OH group, which is the same hydrophilic
group as that of an apatite (Ca10)(PO4)6O) contained in
bones because such a covering layer has affinity with bones, thereby
promoting osteogenesis. A silica gel layer can be formed on the surface
of the bone filling material in the following way. Firstly, acid aqueous
solutions such as hydrochloric acid, nitric acid, and sulfuric acid are
mixed in liquid glass (Na2OSiO2nH2O). And when the mixture
showed a proper viscosity, the bone filling material is impregnated with
the mixture, and then it is taken out from the mixture and is soaked in
water so that Na.sup.+ ions are dissolved in water. The silica gel layer
may also be formed in the following way. Firstly, hydrolysis or
polymerization reaction of alkoxide of silicon (or titanium) is promoted
by mixing alcohol solution of tetramethoxysilane (titanate),
tetraethoxysilane (titanate), tetraproxysilane (titanate),
tetraisopropoxysilane (titanate), or tetrabutoxysilane (titanate), which
are alkoxide of the silicon (or titanium), with catalysers (acid aqueous
solution such as hydrochloric acid, nitric acid, sulfuric acid, and
acetic acid, or ammonia water solution) as needed. When the mixture
showed a proper viscosity, the bone filling material is impregnated with
the mixture, and then it is taken out from the mixture.

Bone Filling Material

[0137]The bone filling material obtained by the above method has high
dimensional accuracy and exhibits less unevenness. Also, there are less
defective materials produced, and mass production of the bone filling
material is made possible by the above method. A preferred embodiment of
the bone filling material of the present invention preferably has a shape
having a plurality of protruding parts. And it is more preferred that the
protruding parts are provided so that they are arranged in linear
symmetry, plane symmetry, and spatial symmetry. A specific preferred
shape of the bone filling material is a tetrapod-shape (a shape having
four protruding parts extending toward each vertex from the center of the
regular tetrahedron), or a shape having n (n=6, 8, 12, etc.) protruding
parts extending toward each vertex from the center of the regular body
having n faces. The size of the bone filling material (the diameter of a
sphere that the bone filling material can be accommodated) is, for
example, from 1×10-2 mm to 5 mm, preferably from
5×10-2 mm to 3 mm, further preferably from 1×10-2
mm to 2 mm, more preferably from 2×10-1 mm to 1.5 mm.
Furthermore, a certain disease can be effectively treated and a proper
dosage of pharmaceutical agent can be administered by using bone filling
material having predetermined pharmaceutical agent administered on its
surface. Hereinafter, preferred shapes of the bone filling material of
the present invention are explained.

[0138]FIG. 2 is a conceptual diagram for explaining an example of a
tetrapod-shaped bone filling material. FIG. 2(A) is a side view, FIG.
2(B) is a top view, and FIG. 2(C) is a perspective view. Note that "Fig."
means "Figure" (the same below). The bone filling material (11) shown in
FIG. 2 is a tetrapod-shaped (a shape having four protruding parts (12)
extending toward each vertex from the center of the regular tetrahedron)
bone filling material. It is preferred that a taper is provided at the
tip part (13) of each protruding part (12) and is smoothly shaped (the
same below). As shown in FIG. 2, each protruding part (12) has
substantially the same shape, but one or two of them may be small shaped.
Also, each protruding part has, for example, a truncated cone shape which
is tapered toward the tip end thereof. The tip portion of each protruding
part may be hemispheric (the same below).

[0139]FIG. 3 is a conceptual diagram for explaining an example of a
tetrapod-shaped bone filling material with its top portion cut off. FIG.
3(A) and FIG. 3(B) are side views, FIG. 3(C) is a top view, and FIG. 3(D)
is a perspective view. The bone filling material according to this
embodiment relates to a tetrapod-shaped bone filling material shown in
FIG. 2 having one protruding part which is cut off in the middle.
Specifically, it is a bone filling material having three protruding parts
extending toward each vertex from the center of the regular tetrahedron,
and a protruding part extending toward the remaining one vertex which is
shorter than the other protruding parts. When a plurality of the above
shaped bone filling material is used, continuous holes of a cluster of
the bone filling material can be made smaller than those of the
tetrapod-shaped bone filling material. So the strength of the whole bone
filling material can be improved.

[0140]FIG. 4 is a conceptual diagram for explaining an example of a bone
filling material having three protruding parts. FIG. 4(A) is a
perspective view, and FIG. 4(B) is a top view. It has protruding parts,
for example, along the ridge lines of the tetrahedron. And the tip part
of each protruding part is, for example, hemispheric. Since the bone
filling material according to this embodiment is entangled with each
other, strongly bonded bone filling material can be formed.

[0141]FIG. 5 is a conceptual diagram for explaining an example of a
hemispherical bone filling material. FIG. 5(A) is a perspective view, and
FIG. 5(B) is a bottom view. The bone filling material according to this
embodiment, as shown in FIG. 5(A), is preferred to have a hole part (21)
penetrating form the top part to the bottom of the hemisphere. When the
bone filling material get together, the hole parts form continuous holes
wherein cells can penetrate easily. The shape of the hole part is not
limited, but as shown in FIG. 5(A), column shape is preferred. When the
radius of the hemisphere is represented by r, the radius of the
column-shaped hole is, for example, from r/10 to 2r/3, and is preferably
from r/5 to r/2. These preferred holes can maintain the strength of the
bone filling material. It is preferred that the bone filling material
according to this embodiment have a mortised part (22) in the
undersurface part. The example of the thickness (23) of the bone filling
material is from r/20 to r/3, and is preferably from r/10 to r/4.

[0142]FIG. 6 is a conceptual diagram for explaining an example of a bone
filling material having one or a plurality of hollowed parts in the
undersurface part. FIG. 6(A) is a perspective view, and FIG. 6(B) is a
bottom view. The bone filling material according to this embodiment, for
example, has one or a plurality of halved column-shaped hollowed parts in
the undersurface part of the hemisphere. It is preferred that the bottom
part of hemisphere and the halved surface of the halved column (plane
parts which is neither the bottom plane nor the top plane) shares the
same plane face. The bone grafting material having these halved hollowed
parts (24) can maintain the strength of the bone grafting material and
have preferred physical properties for culturing cells and the like.
Noted that the bone grafting material described in FIG. 6 can adopt the
composition of the bone filling material described in FIG. 5 as
appropriate. The example of the radius of the circle which is the halved
column-shaped hollowed parts in the undersurface part of the hemisphere
is from r/10 to 2r/3, and is preferably from r/5 to r/2.

[0143]FIG. 7 is a conceptual diagram for explaining an example of a
double-headed-shaped bone filling material. FIG. 7(A) is a perspective
view, and FIG. 7(B) and FIG. 7(C) are side views. The bone filling
material according to this embodiment comprises double-headed parts (32,
33) on both ends of the body part (31). These bone grafting materials
shaped like this entwined with each other, thereby improving the strength
of the whole of the bone grafting materials as well as making the size of
the continuous holes appropriate. FIG. 7 shows an example of a bone
filling material whose head part is connected to the body part in
T-shape, but the head part may be cross-shaped, furthermore a number of
protruding parts may be provided at every predetermined angle. Although,
FIG. 7 showed an example that the two head parts are shifted by 90
degrees, the angle of the two head parts may be 45 degrees, 0 degree, or
30 degree.

[0144]FIG. 8 is a conceptual diagram for explaining an example of a bone
filling material having protruding parts on the center thereof. FIG. 8(A)
is a perspective view, and FIG. 8(B) is a side view. The bone filling
material according to this embodiment is a bone filling material shown in
FIG. 7 further comprising protruding parts which penetrate through the
body part (31). These bone grafting materials shaped like this entwined
with each other, thereby improving the strength of the whole of the bone
grafting materials as well as making the size of the continuous holes
appropriate. The bone filling material according to this embodiment can
adopt the composition of the bone filling material described in FIG. 7 as
appropriate.

[0145]FIG. 9 is a conceptual diagram for explaining an example of a
cross-shaped bone filling material. FIG. 9(A) is a perspective view, FIG.
9(B) is a top view, FIG. 9(C) is a side view, and FIG. 9(D) is a bottom
view. It is preferred that the bone filling material according to this
embodiment, as shown in FIG. 9(C) and FIG. 9(D), has foot part at each
end part of the bottom of the cross. The bone grafting material having
this shape can make the size of the continuous holes appropriate.

[0146]FIG. 10 is a conceptual diagram for explaining an example of a
nearly planar bone filling material. FIG. 10(A) is a perspective view,
FIG. 10(B) is a top view, and FIG. 10(C) is a side view. The bone filling
material according to this embodiment has a hole part in the center of
the bone filling material and groove parts on the side surfaces. The bone
grafting material having this shape can make the size of continuous holes
appropriate.

[0147]FIG. 11 is a conceptual diagram for explaining an example of a bone
filling material having protruding parts on one or both ends of the plane
face. Since the bone filling material according to this embodiment has a
protruding part on the edge part of a flat body part, it cannot simply be
piled up. Also, the bone filling material according to this embodiment
preferably has dent portions in the body part as shown in FIG. 11. The
bone grafting material having this shape can make the size of continuous
holes appropriate.

[0148]FIG. 12 is a conceptual diagram for explaining an example of a bone
filling material having an inclined top surface. FIG. 12(A) is a
perspective view, and FIG. 12(B) is a side view. As shown in FIG. 12, the
bone filling material according to this embodiment comprises a foot part
(42), and a table part (43) mounted on a foot part (42). And the bottom
face of the foot part (42) and the top face of the table part (43) have
inclined structures. The bone grafting material according to this
embodiment having this shape (inclined top face) cannot simply be piled
up, which ensures the existence of continuous holes.

Usage of Bone Filling Material

[0149]The bone filling material produced by the production method of the
present invention is injected in bone defect sites, osteoporosis sites,
or bone elongation sites. Also, in addition to be filled in the gaps of
bones such as bone defect sites, it can also be used as a predetermined
carrier of pharmaceutical agents. In this way, the bone filling material
can be used for treating or preventing not only bone related diseases but
also various other diseases.

Three-Dimensional Cell Culture Carrier Used In Vitro

[0150]Next, a three-dimensional cell culture carrier according to a
preferred aspect of the present invention is explained. The
three-dimensional cell culture carrier basically uses the above described
bone filling material as needed. Namely, the three-dimensional cell
culture carrier of the present invention has a plurality of protruding
parts, and includes a bone filling material containing calcium-based
material. As demonstrated in the example described below, the bone
filling material of the present invention can culture cells
three-dimensionally, because the bone filling material has preferred
voids through which cultured cells can develop. Namely, the present
invention can provide an in vitro cell culture system which is an
administration of the bone filling material.

[0151]It is preferred that the three-dimensional cell culture carrier use
a plurality of the above described bone filling materials. The height of
the three-dimensional cell culture carrier is adjusted in accordance with
the amount of cells to be obtained. The example of the range of the
height is from 1 μm to 1 m, preferably from 3 μm to 10 cm, more
preferably from 10 μm to 5 cm, and further preferably from 50 μm to
1 cm.

[0152]Cells can develop through porous bone filling material effectively.
So, it is preferred that the bone filling material, for example, include
the above described salts as appropriate. A porous bone filling material
can be obtained by immersing the obtained sintering body (which is
obtained by sintering the mixture of ingredient and salt component such
as sodium chloride) in water and dissolving salts. Also, when minute
cracks are made by lowering sinterability, cells can be taken inside the
bone filling material, which enables cell culture preferably.
Sinterability is lowered, for example, by lowering the sintering
temperature or using larger-grained ingredient powders.

[0153]A preferred embodiment of the three-dimensional cell culture carrier
of the present invention is one wherein an adhesiveness-imparting agent
is impregnated or administered on the surface of the bone filling
material. Since the above described three-dimensional cell culture
carrier maintains a preferred steric structure, cell culture can be
continued effectively. The adhesiveness-imparting agent above explained
can be used for the three-dimensional cell culture carrier in the same
manner. The cultured cells may be administered in vivo. Since the above
described adhesiveness-imparting agent has excellent biocompatibility,
the cultured cells can be administered in vivo with little risk to the
human body.

[0154]A usage example of a bone filling material according to a certain
aspect of the present invention is a separating carrier for
chromatography comprising bone filling material including a plurality of
protruding parts and containing calcium-based material. As demonstrated
in the example described below, the bone filling material of the present
invention has preferred sorbability, and the sorbability can be
controlled, for example, by adjusting voids of the bone filling material.
So a plurality of bone filling materials can be used as separating
carriers for chromatography. A specific usage of chromatography is as
follows. Proper amount of bone filling material is filled in a column for
chromatography, and solution is injected in the column.

Example 1

[0155]Hereinafter, a method for producing the bone filling material of the
present invention is explained specifically using an example. However the
present invention is not specifically limited to the example, and
includes modifications, which those skilled in the art can think of.

1. Kneading Step

[0156]α-TCP (produced by Taihei Chemical Industrial, grain size 10
μm) was used as powder ingredient. Binder was contained in amounts of
24 parts by weight with respect to 100 parts by weight of ingredient
powder. Ethylene-vinyl acetate copolymer, polybutylmethacrylate, paraffin
wax, dibutylphthalate and stearic acid were mixed to produce binder. The
blending ratio (weight ratio) thereof was 30:30:30:5:5. A 300 cc pressure
type kneader was heated to 150° C., and binder was poured in the
kneader in the order from the binder having the highest melting point to
the binder having the lowest melting point. And then the mixture was
kneaded for 60 minutes before cooling down. The resultant kneaded
material was crashed in a pot mill made of ceramics, and was used as
material (a compound or pellet) for molding.

2. Molding Step

[0157]A mold was manufactured based on a CAD image of the bone filling
material. FIG. 13 is a CAD generated drawing of a bone filling material.
A twelve-cavity mold made of SKD11 was used. The mold has four protruding
parts. A gate intake is provided on one of them, and mold parting face is
provided along the center line of the remaining three protruding parts
between the fixed side and the movable side as deformed part. Ejector
pins are arranged inside the three protruding parts. A horizontal
injection molding machine (mold locking force: 12 tons) was used. The
initial set value of the injection pressure was set at 12 GPa. The
temperature of the cylinder of the molding machine was set at 130°
C., and the temperature of the mold was set at 20° C.

3. Binder Removal Step

[0158]Molded body was heated to 1000° C. (maximum temperature) from
ambient atmosphere in an atmosphere degreasing furnace, and the
temperature was maintained for an hour before cooling down. The binder
removal step lasted 18 hours including cooling down period. A setter made
of 90% of alumina (porosity 20%) was used.

4. Sintering Step

[0159]A degreased body was heated to 1000° C. (maximum temperature)
from ambient atmosphere, and then the temperature was maintained for an
hour before cooling down. The sintering step lasted 18 hours including
cooling down period. The setter which was used in the binder removal step
was used. The flexural strength of the resultant bone filling material
was 6.1 MPa (n=18).

[0160]The resultant bone filling material is shown in FIG. 14 to 16. FIG.
14 is a photograph, in place of a diagram, showing a bone filling
material obtained in Example 1. FIG. 15 is an electron microgram, in
place of a diagram, showing a bone filling material obtained in Example
1. FIG. 16 is an electron microgram, in place of a diagram, showing
integrated bone filling materials obtained in Example 1.

Test Example 1

Verification of Sintering Temperature of the Bone Filling Material and
Bending Strength Thereof

[0161]Several kinds of bone filling materials were produced by changing
the sintering temperature. And the bending strength of the resultant bone
filling material was verified in accordance with JIS R1601. The result is
shown in FIG. 17. FIG. 17 shows a relationship between the sintering
temperature and the bending strength of the bone filling material
obtained in Example 1. It can be seen form FIG. 17 that the bending
strength of the bone filling material obtained will be higher, as the
sintering temperature thereof becomes higher. It can also be seen that
the bending strength of the resultant bone filling material can be
controlled by adjusting the sintering temperature. It can further be seen
that a bone filling material having bending strength of 2 MPa to 10 MPa
(preferably 4 MPa to 9 MPa, further preferably 6 MPa to 9 MPa) can be
obtained.

Test Example 2

Verification of Filling Condition of the Bone Filling Material In Vivo

[0162]A plurality of bone filling materials form appropriate continuous
holes. And bone regeneration is promoted by biological cells penetrating
into the continuous holes. So it is preferred that the bone filling
material form appropriate continuous holes when it is administered in
vivo. In this test example, verification was made on how the bone filling
materials of the present invention get together. For comparison, it was
also verified whether the existing artificial bone granulation were
assembled. The artificial bone granulation were produced by crushing
block-shaped bone filling material (OSferion produced by Olympus Co.,
Japan). In specific, the structure of continuous holes was confirmed by a
micro X-ray CT when the bone filling material of the present invention
and the existing artificial bone granulation were accumulated in a 2 ml
cryotube. The results are shown in FIG. 18. FIG. 18 are CT images by
microX-rays, in place of a diagram, showing gatherings of the bone
filling materials obtained in Example 1 and the existing artificial bone
product. FIG. 18(A) shows gatherings of the bone filling materials of the
present invention. FIG. 18(B) shows gatherings of the conventional
artificial bone granulation. FIG. 18(C) shows the bone filling materials
of the present invention filled in a cryotube. FIG. 18(D) shows the
existing bone prosthesis products filled in a cryotube.

[0163]It can be seen from FIG. 18 (A) that the bone filling materials of
the present invention tend to be self-organizingly accumulated on the
joint portion of the pods collectively. As a result, the accumulated bone
filling material prevents them from moving relative to each other. It can
also be seen that the above described structure of the continuous holes
form preferred continuous holes. Since the structure of the continuous
holes reflects the length of the pods of the bone filling material and
the angle thereof, the structure can be adjusted by changing the length
and the angle of the pods. So it can be concluded that the bone filling
material of the present invention has excellent morphologic stability and
build bone developing area as appropriate. Furthermore, the CT images
showed that the density of the bone filling material was almost uniform.

[0164]On the other hand, as shown in FIG. 18(B), it is considered that the
artificial bone granulation has uneven inter-grain distance which causes
problems in reproducibility and morphologic stability of the structure of
the continuous holes. In particular, when artificial bone granulations
are embedded in a wounded area which is rich in biological fluid, it is
highly likely that the inter-grain structure thereof collapses. So it is
conceivable that therapeutic effects can not be expected by using the
artificial bone granulations. The artificial bone granulations have air
bubbles of several hundred micron diameter which are contained in the
granulations. When the artificial bone granulations are administered in
vivo, cells are likely to be penetrated into the air bubbles of the
granulations. But the shape of the air bubbles is uneven, which is not
always a preferred shape for promoting osteogenesis. From this
perspective, it can also be considered that a desirable osteogenesis
effect can not be obtained by using the artificial bone granulations.
Also, the density of the existing artificial bone granulations is uneven
having low density parts.

[0165]It can be seen from FIG. 18(C) that the bone filling material of the
present invention has excellent filling property, which makes the
material filled along the wall of the cryotube. In contrast, it can be
seen from FIG. 18(D) that the existing artificial bone products are not
filled along the wall of the cryotube, which may cause voids. (see the
arrow of FIG. 18(D)). These voids cause a collapse of homeostasis when
the artificial bone and the like are embedded. Namely, it is desirable
that proper sized holes or continuous holes are formed when a plurality
of filling materials are get together, but it is not desirable that too
large holes, such as voids, are formed. Thus, it can be seen that the
bone filling material of the present invention act as preferred bone
filling material, when a plurality of them are administered.

Test Example 3

Verification of the Bone Filling Material as Three Dimensional Cell
Culture

[0166]Osteoblast-like cell line MC3T3 (MC3T3-E1 (mouse calvarium-derived
osteoblast-like cell lines)) was plane cultured, and when it became
confluent, the bone filling material unit obtained in Example 1 was
placed thereon. The cell culture was continued, and the unit was observed
with a microscope over time. The culture medium was a Dullbeco's Eagle's
modified medium (DEM media) wherein 10% FBS and 1%
penicillin-streptomycin was added. The medium was replaced once every
four days. To make a comparison, an observation was made on bone filling
material which was set on a media wherein the cell line was not added.
Alkaline Phosphatase (ALP) was stained on the osteoblasts in six days and
ten days. It was confirmed that the bone filling material had a preferred
cellular adhesiveness. The results are shown in FIG. 19.

[0167]FIG. 19(A) to (B) are photographs, in place of diagrams, examining
cellular adhesiveness of a bone filling material of the present
invention. FIG. 19 (A) is a photograph showing a bone filling material
without cultured cells after four days culture. FIG. 19 (B) is a
photograph, after four days culture, showing a bone filling material on
which osteoblast-like cell lines MC3T3 were cultured. FIG. 19 (C) is a
photograph showing a bone filling material without cultured cells which
is Alkaline Phosphatase (ALP) stained after six days culture. FIG. 19 (D)
is a photograph, Alkaline Phosphatase (ALP) stained after six days
culture, showing a bone filling material on which osteoblast-like cell
lines MC3T3 were cultured. FIG. 19 (E) is a photograph showing a bone
filling material without cultured cells which is Alkaline Phosphatase
(ALP) stained after ten days culture. FIG. 19 (F) is a photograph,
Alkaline Phosphatase (ALP) stained after ten days culture, showing a bone
filling material on which osteoblast-like cell lines MC3T3 were cultured.

[0168]It can be seen from FIGS. 19(A) and 19(B) that there are no cells
attached on the surface of the bone filling material set on the medium
without cell lines, but there are cells attached on the surface of the
bone filling material set on the medium with cell lines. The photograph
showing Alkaline Phosphatase (ALP) stained bone filling material after
six days culture (shown in FIG. 19(D)) shows how the osteoblasts climb
the side gradient of the bone filling material. It can be seen from FIG.
19(F) that there are cells attached on all over the surface of the bone
filling material which was cultured for ten days. Therefore, it can also
be seen that the bone filling material of the present invention acts
effectively as a three-dimensional culture carrier.

[0169]The bone filling material produced by the production method of the
present invention is injected in bone defect sites, osteoporosis sites,
or bone elongation sites. Also, in addition to be filled in the gaps of
bones such as bone defect sites, it can also be used as a predetermined
carrier of pharmaceutical agents. So the bone filling material of the
present invention can be used in the field of pharmaceutical industry and
the like.

[0170]Cell culture has been so far performed two-dimensionally. But, in
contrast, the present invention can provide a carrier which can perform
cell culture three-dimensionally. So it can be used in the field of
pharmaceutical and biotechnology industries in which cell culture is
performed.